Distinct intron DNA structures in simian virus 40 T-antigen and adenovirus 2 E1A genes

Distinct structures delineating the introns of Simian Virus 40 T-antigen and Adenovirus 2 E1A genes have been discovered. The structures, which are centered around the branch points of the genes inserted in supercoiled double-stranded plasmids, are specifically targeted through photoactivated strand cleavage by the metal complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III). The DNA sites that are recognized lack sequence homology but are similar in demarcating functionally important sites on the RNA level. The single-stranded DNA fragments corresponding to the coding strands of the genes were also found to fold into a structure apparently identical to that in the supercoiled genes based on the recognition by the metal complex. Further investigation of different single-stranded DNA fragments with other structural probes, such as another metal complex bis(1,10-phenanthroline)(phenanthrenequinone diimine)rhodium(III), AMT (4'aminomethyl-4,5',8 trimethylpsoralen), restriction enzyme Mse I, and mung bean nuclease, showed that the structures require the sequ ences at both ends of the intron plus the flanking sequences but not the middle of the intron. The two ends form independent helices which interact with each other to form the global tertiary structures. Both of the intron structures share similarities to the structure of the Holliday junction, which is also known to be specifically targeted by the former metal complex. These structures may have arisen from early RNA intron structures and may have been used to facilitate the evolution of genes through exon shuffling by acting as target sites for recombinase enzymes.

I. Rigid body penetration into brittle materials. II. Phase change effect on shock wave propagation

Part I.

We have developed a technique for measuring the depth time history of rigid body penetration into brittle materials (hard rocks and concretes) under a deceleration of ~ 10⁵ g. The technique includes bar-coded projectile, sabot-projectile separation, detection and recording systems. Because the technique can give very dense data on penetration depth time history, penetration velocity can be deduced. Error analysis shows that the technique has a small intrinsic error of ~ 3-4 % in time during penetration, and 0.3 to 0.7 mm in penetration depth. A series of 4140 steel projectile penetration into G-mixture mortar targets have been conducted using the Caltech 40 mm gas/ powder gun in the velocity range of 100 to 500 m/s.

We report, for the first time, the whole depth-time history of rigid body penetration into brittle materials (the G-mixture mortar) under 10⁵ g deceleration. Based on the experimental results, including penetration depth time history, damage of recovered target and projectile materials and theoretical analysis, we find:

1. Target materials are damaged via compacting in the region in front of a projectile and via brittle radial and lateral crack propagation in the region surrounding the penetration path. The results suggest that expected cracks in front of penetrators may be stopped by a comminuted region that is induced by wave propagation. Aggregate erosion on the projectile lateral surface is < 20% of the final penetration depth. This result suggests that the effect of lateral friction on the penetration process can be ignored.

2. Final penetration depth, P_max, is linearly scaled with initial projectile energy per unit cross-section area, e_s , when targets are intact after impact. Based on the experimental data on the mortar targets, the relation is P_max(mm) 1.15e_s (J/mm² ) + 16.39.

3. Estimation of the energy needed to create an unit penetration volume suggests that the average pressure acting on the target material during penetration is ~ 10 to 20 times higher than the unconfined strength of target materials under quasi-static loading, and 3 to 4 times higher than the possible highest pressure due to friction and material strength and its rate dependence. In addition, the experimental data show that the interaction between cracks and the target free surface significantly affects the penetration process.

4. Based on the fact that the penetration duration, t_max, increases slowly with e_s and does not depend on projectile radius approximately, the dependence of t_max on projectile length is suggested to be described by t_max(μs) = 2.08e_s (J/mm² + 349.0 x m/(πR²), in which m is the projectile mass in grams and R is the projectile radius in mm. The prediction from this relation is in reasonable agreement with the experimental data for different projectile lengths.

5. Deduced penetration velocity time histories suggest that whole penetration history is divided into three stages: (1) An initial stage in which the projectile velocity change is small due to very small contact area between the projectile and target materials; (2) A steady penetration stage in which projectile velocity continues to decrease smoothly; (3) A penetration stop stage in which projectile deceleration jumps up when velocities are close to a critical value of ~ 35 m/s.

6. Deduced averaged deceleration, a, in the steady penetration stage for projectiles with same dimensions is found to be a(g) = 192.4v + 1.89 x 10⁴, where v is initial projectile velocity in m/s. The average pressure acting on target materials during penetration is estimated to be very comparable to shock wave pressure.

7. A similarity of penetration process is found to be described by a relation between normalized penetration depth, P/P_max, and normalized penetration time, t/t_max, as P/P_max = f(t/t_max, where f is a function of t/t_max. After f(t/t_max is determined using experimental data for projectiles with 150 mm length, the penetration depth time history for projectiles with 100 mm length predicted by this relation is in good agreement with experimental data. This similarity also predicts that average deceleration increases with decreasing projectile length, that is verified by the experimental data.

8. Based on the penetration process analysis and the present data, a first principle model for rigid body penetration is suggested. The model incorporates the models for contact area between projectile and target materials, friction coefficient, penetration stop criterion, and normal stress on the projectile surface. The most important assumptions used in the model are: (1) The penetration process can be treated as a series of impact events, therefore, pressure normal to projectile surface is estimated using the Hugoniot relation of target material; (2) The necessary condition for penetration is that the pressure acting on target materials is not lower than the Hugoniot elastic limit; (3) The friction force on projectile lateral surface can be ignored due to cavitation during penetration. All the parameters involved in the model are determined based on independent experimental data. The penetration depth time histories predicted from the model are in good agreement with the experimental data.

9. Based on planar impact and previous quasi-static experimental data, the strain rate dependence of the mortar compressive strength is described by σ_f/σ⁰_f = exp(0.0905(log(έ/έ_0) ^1.14, in the strain rate range of 10^-7/s to 10³/s (σ⁰_f and έ are reference compressive strength and strain rate, respectively). The non-dispersive Hugoniot elastic wave in the G-mixture has an amplitude of ~ 0.14 GPa and a velocity of ~ 4.3 km/s.

Part II.

Stress wave profiles in vitreous GeO₂ were measured using piezoresistance gauges in the pressure range of 5 to 18 GPa under planar plate and spherical projectile impact. Experimental data show that the response of vitreous GeO₂ to planar shock loading can be divided into three stages: (1) A ramp elastic precursor has peak amplitude of 4 GPa and peak particle velocity of 333 m/s. Wave velocity decreases from initial longitudinal elastic wave velocity of 3.5 km/s to 2.9 km/s at 4 GPa; (2) A ramp wave with amplitude of 2.11 GPa follows the precursor when peak loading pressure is 8.4 GPa. Wave velocity drops to the value below bulk wave velocity in this stage; (3) A shock wave achieving final shock state forms when peak pressure is > 6 GPa. The Hugoniot relation is D = 0.917 + 1.711u (km/s) using present data and the data of Jackson and Ahrens [1979] when shock wave pressure is between 6 and 40 GPa for ρ₀ = 3.655 gj cm³ . Based on the present data, the phase change from 4-fold to 6-fold coordination of Ge⁺⁴ with O^-2 in vitreous GeO₂ occurs in the pressure range of 4 to 15 ± 1 GPa under planar shock loading. Comparison of the shock loading data for fused SiO₂ to that on vitreous GeO₂ demonstrates that transformation to the rutile structure in both media are similar. The Hugoniots of vitreous GeO₂ and fused SiO₂ are found to coincide approximately if pressure in fused SiO₂ is scaled by the ratio of fused SiO₂to vitreous GeO₂ density. This result, as well as the same structure, provides the basis for considering vitreous Ge0₂ as an analogous material to fused SiO₂ under shock loading. Experimental results from the spherical projectile impact demonstrate: (1) The supported elastic shock in fused SiO₂ decays less rapidly than a linear elastic wave when elastic wave stress amplitude is higher than 4 GPa. The supported elastic shock in vitreous GeO₂ decays faster than a linear elastic wave; (2) In vitreous GeO₂ , unsupported shock waves decays with peak pressure in the phase transition range (4-15 GPa) with propagation distance, x, as α 1/x^-3.35 , close to the prediction of Chen et al. [1998]. Based on a simple analysis on spherical wave propagation, we find that the different decay rates of a spherical elastic wave in fused SiO₂ and vitreous GeO₂ is predictable on the base of the compressibility variation with stress under one-dimensional strain condition in the two materials.

Synthesis, structure, and photophysics of polypyridophenazine transition-metal complexes

The condensation of phenanthroline-5,6-dione (phendione) with polyamines is a versatile synthetic route to a wide variety of chelating ligands. Condensation with 2,3- napthalene diamine gives benzo[i]dipyrido[3,2-a:2',3'-c]phenazine (bdppz) a ligand containing weakly-coupled orbitals of benzophenazine (bpz) and 2,2' -bipyridinde(bpy) character. The bpy character gives Re and Ru complexes excited-state redox properties; intramolecular electron transfer (ET) takes place to the bpz portion of the ligand. The charge-separated state so produced has an extraordinarily-long 50 µs lifetime. The slow rate of charge recombination arises from a combination of extremely weak coupling between the metal center and the bpz acceptor orbital and Marcus "inverted region" behavior. Molecular orbital calculations show that only 3% the electron density in the lowest unoccupied molecular orbital lies on the bpy atoms of bdppz, effectively trapping the transferred electron on the bpz portion. The rate of charge recombination decreases with increasing driving force, showing that these rates lie in the inverted region. Comparison of forward and back ET rates shows that donor-acceptor coupling is four orders of magnitude greater for photoinduced electron transfer than it is for thermal charge recombination.

Condensation of phendione with itself or tetramines gives a series of binucleating tetrapyridophenazine ligands of incrementally-varying coordination-site separation. When a photoredox-active metal center is attached, excited-state energy and electron transfer to an acceptor metal center at the other coordination site can be studied as a function of distance. A variety of monometallic and homo- and heterodimetallic tetrapyridophenazine complexes has been synthesized. Electro- and magnetochemistry show that no ground-state interaction exists between the metals in bimetallic complexes. Excited-state energy and electron transfer, however, takes place at rates which are invariant with increasing donor-acceptor separation, indicating that a very efficient coupling mechanism is at work. Theory and experiment have suggested that such behavior might exist in extended π-systems like those presented by these ligands.

Condensation of three equivalents of 4,5-dimethyl-1,2-phenylenediamine with hexaketocyclohexane gives the trinucleating ligand hexaazahexamethyltrinapthalene (hhtn). Attaching two photredox-active metal centers and a third catalytic center to hhtn provides means by which multielectron photocatalyzed reactions might be carried out. The coordination properties of hhtn have been examined; X-ray crystallographic structure determination shows that the ligand's constricted coordination pocket leads to distorted geometries in its mono- and dimetallic derivatives.

Engineering multi-step electron tunneling systems in proteins

Multi-step electron tunneling, or “hopping,” has become a fast-developing research field with studies ranging from theoretical modeling systems, inorganic complexes, to biological systems. In particular, the field is exploring hopping mechanisms in new proteins and protein complexes, as well as further understanding the classical biological hopping systems such as ribonuclease reductase, DNA photolyases, and photosystem II. Despite the plethora of natural systems, only a few biologically engineered systems exist. Engineered hopping systems can provide valuable information on key structural and electronic features, just like other kinds of biological model systems. Also, engineered systems can harness common biologic processes and utilize them for alternative reactions. In this thesis, two new hopping systems are engineered and characterized.

The protein Pseudomonas aeruginosa azurin is used as a building block to create the two new hopping systems. Besides being well studied and amenable to mutation, azurin already has been used to successfully engineer a hopping system. The two hopping systems presented in this thesis have a histidine-attached high potential rhenium 4,7-dimethyl-1,10-phenanthroline tricarbonyl [Re(dmp)(CO)3] + label which, when excited, acts as the initial electron acceptor. The metal donor is the type I copper of the azurin protein. The hopping intermediates are all tryptophan, an amino acid mutated into the azurin at select sites between the photoactive metal label and the protein metal site. One system exhibits an inter-molecular hopping through a protein dimer interface; the other system undergoes intra-molecular multi-hopping utilizing a tryptophan “wire.” The electron transfer reactions are triggered by excitation of the rhenium label and monitored by UV-Visible transient absorption, luminescence decays measurements, and time-resolved Infrared spectroscopy (TRIR). Both systems were structurally characterized by protein X-ray crystallography.

Proximity effect in Ag-Pb alloys

The superconducting properties and the microstructure of the Ag_100-xPb_x alloys, 1 ≤ x ≤ 5, prepared by rapid quenching from the liquid state with and without subsequent heat treatments, have been studied. The x-ray diffraction measurements show that supersaturated solid solutions of Pb in Ag can be obtained up to 3.2 at.% Pb as compared to less than 0.1 at.% Pb at equilibrium. It was found that by suitable heat treatment it is possible to vary the size and distribution of the Pb precipitates in the Ag matrix and reproducible superconducting properties in the alloy can be observed. The superconducting transition temperature of these samples can be qualitatively explained by the Silvert and Singh's theoretical calculation. The theory developed for the case of layer structure can be extended to three dimensions to explain the critical current versus temperature behavior. The critical current versus field behavior of these alloys can be explained by the modification of the Josephson effect. Combining these results together with the critical magnetic field measurements and the microstructure studies of the alloys, it can be concluded that the three-dimensional proximity effect is the main mechanism for the superconductivity in the Ag-Pb alloys. Based on the Hilsch empirical formula which was based on experimental results obtained on layer structures, the experimental data in this investigation show that the electron-phonon-electron interaction in silver is attractive. The interaction parameter NV obtained is approximately 0.06, which would lead to a value of 10^-5 °K for the superconducting transition temperature of Ag. These values are in agreement with other determinations which were done on vapor-deposited metallic film sandwiches. Hence, the Hilsch empirical relation valid for layer structures is also valid in the three-dimensional case. Because the transition temperature and the critical current can be varied in a wide range by controlling the heat treatments, the Ag-Pb superconductors might have some useful applications.

Nanofabrication for On-Chip Optical Levitation, Atom-Trapping, and Superconducting Quantum Circuits

Researchers have spent decades refining and improving their methods for fabricating smaller, finer-tuned, higher-quality nanoscale optical elements with the goal of making more sensitive and accurate measurements of the world around them using optics. Quantum optics has been a well-established tool of choice in making these increasingly sensitive measurements which have repeatedly pushed the limits on the accuracy of measurement set forth by quantum mechanics. A recent development in quantum optics has been a creative integration of robust, high-quality, and well-established macroscopic experimental systems with highly-engineerable on-chip nanoscale oscillators fabricated in cleanrooms. However, merging large systems with nanoscale oscillators often require them to have extremely high aspect-ratios, which make them extremely delicate and difficult to fabricate with an "experimentally reasonable" repeatability, yield and high quality. In this work we give an overview of our research, which focused on microscopic oscillators which are coupled with macroscopic optical cavities towards the goal of cooling them to their motional ground state in room temperature environments. The quality factor of a mechanical resonator is an important figure of merit for various sensing applications and observing quantum behavior. We demonstrated a technique for pushing the quality factor of a micromechanical resonator beyond conventional material and fabrication limits by using an optical field to stiffen and trap a particular motional mode of a nanoscale oscillator. Optical forces increase the oscillation frequency by storing most of the mechanical energy in a nearly loss-less optical potential, thereby strongly diluting the effects of material dissipation. By placing a 130 nm thick SiO₂ pendulum in an optical standing wave, we achieve an increase in the pendulum center-of-mass frequency from 6.2 to 145 kHz. The corresponding quality factor increases 50-fold from its intrinsic value to a final value of Q_m = 5.8(1.1) x 10⁵, representing more than an order of magnitude improvement over the conventional limits of SiO₂ for a pendulum geometry. Our technique may enable new opportunities for mechanical sensing and facilitate observations of quantum behavior in this class of mechanical systems. We then give a detailed overview of the techniques used to produce high-aspect-ratio nanostructures with applications in a wide range of quantum optics experiments. The ability to fabricate such nanodevices with high precision opens the door to a vast array of experiments which integrate macroscopic optical setups with lithographically engineered nanodevices. Coupled with atom-trapping experiments in the Kimble Lab, we use these techniques to realize a new waveguide chip designed to address ultra-cold atoms along lithographically patterned nanobeams which have large atom-photon coupling and near 4π Steradian optical access for cooling and trapping atoms. We describe a fully integrated and scalable design where cold atoms are spatially overlapped with the nanostring cavities in order to observe a resonant optical depth of d₀ ≈ 0.15. The nanodevice illuminates new possibilities for integrating atoms into photonic circuits and engineering quantum states of atoms and light on a microscopic scale. We then describe our work with superconducting microwave resonators coupled to a phononic cavity towards the goal of building an integrated device for quantum-limited microwave-to-optical wavelength conversion. We give an overview of our characterizations of several types of substrates for fabricating a low-loss high-frequency electromechanical system. We describe our electromechanical system fabricated on a Si₃N₄ membrane which consists of a 12 GHz superconducting LC resonator coupled capacitively to the high frequency localized modes of a phononic nanobeam. Using our suspended membrane geometry we isolate our system from substrates with significant loss tangents, drastically reducing the parasitic capacitance of our superconducting circuit to ≈ 2.5$ fF. This opens up a number of possibilities in making a new class of low-loss high-frequency electromechanics with relatively large electromechanical coupling. We present our substrate studies, fabrication methods, and device characterization.

High pressure states in condensed matter: I. High pressure behavior of the iron-sulphur system with applications to the earth's core. II. Empirical equation of state for organic compounds at high pressures

Part I:

The earth's core is generally accepted to be composed primarily of iron, with an admixture of other elements. Because the outer core is observed not to transmit shear waves at seismic frequencies, it is known to be liquid or primarily liquid. A new equation of state is presented for liquid iron, in the form of parameters for the 4th order Birch-Murnaghan and Mie-Grüneisen equations of state. The parameters were constrained by a set of values for numerous properties compiled from the literature. A detailed theoretical model is used to constrain the P-T behavior of the heat capacity, based on recent advances in the understanding of the interatomic potentials for transition metals. At the reference pressure of 10⁵ Pa and temperature of 1811 K (the normal melting point of Fe), the parameters are: ρ = 7037 kg/m³, K_S0 = 110 GPa, K_S' = 4.53, K_S" = -.0337 GPa-1, and γ = 2.8, with γ α ρ^-1.17. Comparison of the properties predicted by this model with the earth model PREM indicates that the outer core is 8 to 10 % less dense than pure liquid Fe at the same conditions. The inner core is also found to be 3 to 5% less dense than pure liquid Fe, supporting the idea of a partially molten inner core. The density deficit of the outer core implies that the elements dissolved in the liquid Fe are predominantly of lower atomic weight than Fe. Of the candidate light elements favored by researchers, only sulfur readily dissolves into Fe at low pressure, which means that this element was almost certainly concentrated in the core at early times. New melting data are presented for FeS and FeS₂ which indicate that the FeS₂ is the S-hearing liquidus solid phase at inner core pressures. Consideration of the requirement that the inner core boundary be observable by seismological means and the freezing behavior of solutions leads to the possibility that the outer core may contain a significant fraction of solid material. It is found that convection in the outer core is not hindered if the solid particles are entrained in the fluid flow. This model for a core of Fe and S admits temperatures in the range 3450K to 4200K at the top of the core. An all liquid Fe-S outer core would require a temperature of about 4900 K at the top of the core.

Part II.

The abundance of uses for organic compounds in the modern world results in many applications in which these materials are subjected to high pressures. This leads to the desire to be able to describe the behavior of these materials under such conditions. Unfortunately, the number of compounds is much greater than the number of experimental data available for many of the important properties. In the past, one approach that has worked well is the calculation of appropriate properties by summing the contributions from the organic functional groups making up molecules of the compounds in question. A new set of group contributions for the molar volume, volume thermal expansivity, heat capacity, and the Rao function is presented for functional groups containing C, H, and O. This set is, in most cases, limited in application to low molecular liquids. A new technique for the calculation of the pressure derivative of the bulk modulus is also presented. Comparison with data indicates that the presented technique works very well for most low molecular hydrocarbon liquids and somewhat less well for oxygen-bearing compounds. A similar comparison of previous results for polymers indicates that the existing tabulations of group contributions for this class of materials is in need of revision. There is also evidence that the Rao function contributions for polymers and low molecular compounds are somewhat different.

Electron transfer in chemically and genetically modified myoglobins

The temperature dependences of the reduction potentials (E^o') of wildtype human myoglobin (Mb) and three site-directed mutants have been measured by using thin-layer spectroelectrochemistry. Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn. At pH 7.0, reduction of the heme iron (III) in the former two proteins is accompanied by uptake of a proton by the protein. The changes in E^o', and the standard entropy (ΔS^o') and enthalpy (ΔH^o') of reduction in the mutant proteins were determined relative to values for wild-type; the change in E^o' at 25°C was about -200 millivolts for the Glu and Asp mutants, and about -80 millivolts for the Asn mutant. Reduction of Fe(III) to Fe(II) in the Glu and Asp mutants is accompanied by uptake of a proton. These studies demonstrate that Mb can tolerate substitution of a buried hydrophobic group by potentially charged and polar residues, and that such amino acid replacements can lead to substantial changes in the redox thermodynamics of the protein.

Through analysis of the temperature dependence and shapes of NMR dispersion signals, it is determined that a water molecule is bound to the sixth coordination site of the ferric heme in the Val68Asp and in the Val68Asn recombinant proteins while the carboxyl group of the sidechain of Glu68 occupies this position in Val68Glu. The relative rhombic distortions in the ESR spectra of these mutant proteins combined with H₂¹⁷O and spin interconversion experiments performed on them confirm the conclusions of the NMRD study.

The rates of intramolecular electron transfer (ET) of (NH₃)₅Ru-His48 (Val68Asp, His81GIn, Cys110AIa)Mb and (NH₃)₅Ru-His48 (Val68GIu,His81GIn,Cys110Ala)Mb were measured to be .85(3)s^-1 and .30(2)s^-1, respectively. This data supports the hypothesis that entropy of 111 reduction and reorganization energy of ET are inversely related. The rates of forward and reverse ET for (NH₃)₅ Ru-His48 (Val68GIu, His81 GIn, Cys110AIa)ZnMb -7.2(5)•10⁴s^-1and 1.4(2)•10⁵s^-1, respectively- demonstrate that the placement of a highly polar residue nearby does not significantly change the reorganization energy of the photoactive Zn porphyrin.

The distal histidine imidazoles of (NH₃)₄isnRu-His48 SWMb and (NH₃)₅Ru-His48 SWMb were cyanated with BrCN. The intramolecular ET rates of these BrCN-modified Mb derivatives are 5.5(6)s^-1 and 3.2(5)s^-1, respectively. These respective rates are 20 and 10 times faster than those of their noncyanated counterparts after the differences in ET rate from driving force are scaled according to the Marcus equation. This increase in ET rate of the cyanated Mb derivatives is attributed to lower reorganization energy since the cyanated Mb heme is pentacoordinate in both oxidation states; whereas, the native Mb heme loses a water molecule upon reduction so that it changes from six to five coordinate. The reorganization energy from Fe-OH₂ dissociation is estimated to be .2eV. This conclusion is used to reconcile data from previous experiments in our lab. ET in photoactive porphyrin-substituted myoglobins proceed faster than predicted by Marcus Theory when it is assumed that the only difference in ET parameters between photoactive porphyrins and native heme systems is driving force. However, the data can be consistently fit to Marcus Theory if one corrects for the smaller reorganization in the photoactive porphyrin systems since they do not undergo a coordination change upon ET.

Finally, the intramolecular ET rate of (NH₃)₄isnRu-His48 SWMb was measured to be 3.0(4)s^-1. This rate is within experimental error of that for (NH₃)₄pyrRu-His48 SWMb even though the former has 80mV more driving force. One likely possibility for this observation is that the tetraamminepyridineruthenium group undergoes less reorganization upon ET than the tetraammineisonicotinamideruthenium group. Moreover, analysis of the (NH₃)₄isnRu-His48 SWMb experimental system gives a likely explanation of why ET was not observed previously in (NH₃)₄isnRu-Cytochrome C.

I. Thermochemistry and reaction kinetics of disolvated protons by ion cyclotron resonance spectroscopy. II. Thermochemical studies of small fluorocarbons by photoionization mass spectrometry

The disolvated proton, H(OH₂)₂⁺ is employed as a chemical reagent in low pressure (˂ 10^-5 torr) investigations by ion cyclotron resonance spectroscopy. Since termolecular reactions are absent at low pressure, disolvated protons are not generally observed. However H(OH₂)₂⁺ is produced in a sequence of bimolecular reactions in mixtures containing H₂O and one of a small number of organohalide precursors. Then a series of hydrated Lewis bases is produced by H₃O⁺ transfer from H(OH₂)₂⁺. In Chapter II, the relative stability of hydrated bases containing heteroatoms of both first and second row elements is determined from the preferred direction of H₃O⁺ transfer between BH(OH₂)⁺ complexes. S and P containing bases are shown to bind H₃O⁺ more weakly than O and N bases with comparable proton affinities. A simple model of hydrogen bonding is proposed to account for these observations.

H⁺ transfer from H(OH₂)₂⁺ to several Lewis bases also occurs at low pressure. In Chapter III the relative importance of H₃O⁺ transfer and H⁺ transfer from H(OH₂)₂⁺ to a series of bases is observed to be a function of base strength. Beginning with CH₃COOH, the weakest base for which H⁺ transfer is observed, the importance of H⁺ transfer increases with increasing proton affinity of the acceptor base. The nature of neutral products formed from H(OH₂)₂⁺ by loss of H⁺ is also considered.

Chapters IV and V deal with thermochemistry of small fluorocarbons determined by photoionization mass spectrometry. The enthalpy of formation of CF₂ is considered in Chapter IV. Photoionization of perfluoropropylene, perfluorocyclopropane, and trifluoromethyl benzene yield onsets for ions formed by loss of a CF₂ neutral fragment. Earlier determinations of ΔH^°_f298 (CF₂) are reinterpreted using updated thermochemical values and compared with results of this study. The heat of formation of neutral perfluorocyclopropane is also derived. Finally, the energetics of interconversion of perfluoropropylene and perfluorocyclopropane are considered for both the neutrals and their molecular ions.

In Chapter V the heats of formation of CF₃⁺ and CF₃I⁺are derived from photoionization of CF₃I. These are considered with respect to ion-molecule reactions observed in CF₃I monitored by the techniques of ion cyclotron resonance spectroscopy. Results obtained in previous experiments are also compared.

Studies on 4S and 5S RNA of HeLa cells

SECTION I

Section I is concerned with a partial sequence analysis conducted on 5S RNA from HeLa cells. Analysis of the oligonucleotide pattern after pancreatic ribonuclease digestion of a highly-purified preparation of 5S RNA gave results which were in general agreement with those published for KB cells, both with respect to the identity and the frequency of the partial sequences. However, the presence of a trinucleotide not found in the KB 5S pattern, together with the reproducibly much lower than expected molar yield of the larger oligonucleotides strongly suggested the occurrence of alternate sequences at various sites in the 5S molecules of human cells. The presence of ppGp and pppGp at the 5'-terminus of HeLa 5S RNA was clearly demonstrated. The implications of this finding with regard to the origin of 5S RNA are discussed.

SECTION II

In Section II the proportion of the HeLa cell genome complementary to tRNA was investigated by using RNA- DNA hybridization. The value for saturation of the HeLa DNA by tRNA was found to be 1.1 x 10^-5, which corresponds to about 4900 sites for tRNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized tRNA revealed significant differences from the nucleotide composition of the input tRNA, with the purine to pyrimidine ratio indicating, however, that these differences were not produced by excessive RNase attack of the hybrid. The size of the hybridized tRNA was only moderately smaller than that of the input RNA; the average S value in formaldehyde was 2.7 (corresponding to a length of about 65 nucleotides), suggesting that a relatively small portion near the ends of the hybridized 4S chains had been removed by RNase.

SECTION III

The proportion of the HeLa cell genome complementary to 5S RNA was investigated by using RNA-DNA hybridization. The value for saturation of the HeLa DNA by 5S RNA was found to be 2.3 x 10^-5, which corresponds to about 7,000 sites for 5S RNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized 5S RNA revealed no significant difference from the nucleotide composition of the input RNA. At the RNA to DNA input ratio of 1:1000, the average S value in formaldehyde of the hybridized 5S RNA corresponded to a polynucleotide chain about two-thirds the size of the input RNA.

I. The rate and mechanism of the vapor-phase reaction between water and parts-per-million concentrations of nitrogen dioxide. II. The rate and mechanism of the air oxidation of parts-per-million concentrations of nitric oxide in the presence of water vapor.

Part I

A study of the thermal reaction of water vapor and parts-per-million concentrations of nitrogen dioxide was carried out at ambient temperature and at atmospheric pressure. Nitric oxide and nitric acid vapor were the principal products. The initial rate of disappearance of nitrogen dioxide was first order with respect to water vapor and second order with respect to nitrogen dioxide. An initial third-order rate constant of 5.5 (± 0.29) x 10⁴ liter² mole^-2 sec^-1 was found at 25˚C. The rate of reaction decreased with increasing temperature. In the temperature range of 25˚C to 50˚C, an activation energy of -978 (± 20) calories was found.

The reaction did not go to completion. From measurements as the reaction approached equilibrium, the free energy of nitric acid vapor was calculated. This value was -18.58 (± 0.04) kilocalories at 25˚C.

The initial rate of reaction was unaffected by the presence of oxygen and was retarded by the presence of nitric oxide. There were no appreciable effects due to the surface of the reactor. Nitric oxide and nitrogen dioxide were monitored by gas chromatography during the reaction.

Part II

The air oxidation of nitric oxide, and the oxidation of nitric oxide in the presence of water vapor, were studied in a glass reactor at ambient temperatures and at atmospheric pressure. The concentration of nitric oxide was less than 100 parts-per-million. The concentration of nitrogen dioxide was monitored by gas chromatography during the reaction.

For the dry oxidation, the third-order rate constant was 1.46 (± 0.03) x 10⁴ liter² mole^-2 sec^-1 at 25˚C. The activation energy, obtained from measurements between 25˚C and 50˚C, was -1.197 (±0.02) kilocalories.

The presence of water vapor during the oxidation caused the formation of nitrous acid vapor when nitric oxide, nitrogen dioxide and water vapor combined. By measuring the difference between the concentrations of nitrogen dioxide during the wet and dry oxidations, the rate of formation of nitrous acid vapor was found. The third-order rate constant for the formation of nitrous acid vapor was equal to 1.5 (± 0.5) x 10⁵ liter² mole^-2 sec^-1 at 40˚C. The reaction rate did not change measurably when the temperature was increased to 50˚C. The formation of nitric acid vapor was prevented by keeping the concentration of nitrogen dioxide low.

Surface effects were appreciable for the wet tests. Below 35˚C, the rate of appearance of nitrogen dioxide increased with increasing surface. Above 40˚C, the effect of surface was small.

The instability of fluids with time dependent heating

The stability of a fluid having a non-uniform temperature stratification is examined analytically for the response of infinitesimal disturbances. The growth rates of disturbances have been established for a semi-infinite fluid for Rayleigh numbers of 10³, 10⁴, and 10⁵ and for Prandtl numbers of 7.0 and 0.7.

The critical Rayleigh number for a semi-infinite fluid, based on the effective fluid depth, is found to be 32, while it is shown that for a finite fluid layer the critical Rayleigh number depends on the rate of heating. The minimum critical Rayleigh number, based on the depth of a fluid layer, is found to be 1340.

The stability of a finite fluid layer is examined for two special forms of heating. The first is constant flux heating, while in the second, the temperature of the lower surface is increased uniformly in time. In both cases, it is shown that for moderate rates of heating the critical Rayleigh number is reduced, over the value for very slow heating, while for very rapid heating the critical Rayleigh number is greatly increased. These results agree with published experimental observations.

The question of steady, non-cellular convection is given qualitative consideration. It is concluded that, although the motion may originate from infinitesimal disturbances during non-uniform heating, the final flow field is intrinsically non-linear.

Nuclear reactions leading to energy levels of Ar37 and K37

In view of recent interest in the Cl³⁷ (ʋ solar’^e-)Ar³⁷ reaction cross section, information on some aspects of mass 37 nuclei has been obtained using the K³⁹ (d, ∝) Ar³⁷ and Cl³⁵ (He³, p) Ar³⁷ reactions. Ar³⁷ levels have been found at 0, 1.41, 1.62, 2.22, 2.50, 2.80, 3.17, 3.27, 3.53, 3.61, 3.71, (3.75), (3.90), 3.94, 4.02, (4.21), 4.28, 4.32, 4.40, 4.45, 4.58, 4.63, 4.74, 4.89, 4.98, 5.05, 5.10, 5.13, 5.21, 5.35, 5.41, 5.44, 5.54, 5.58, 5.67, 5.77, and 5.85 MeV (the underlined values correspond to previously tabulated levels). The nuclear temperature calculated from the Ar³⁷ level density is 1.4 MeV. Angular distributions of the lowest six levels with the K³⁹ (d, ∝) Ar³⁷ reaction at E_d = 10 MeV indicate a dominant direct interaction mechanism and the inapplicability of the 2I + 1 rule of the statistical model. Comparison of the spectra obtained with the K³⁹ (d, ∝) Ar³⁷ and Cl³⁵ (He³, p) Ar³⁷ reactions leads to the suggestion that the 5.13-MeV level is the T = 3/2 Cl³⁷ ground state analog. The ground state Q-value of the Ca⁴⁰ (p, ∝) K³⁷ reaction has been measured: -5179 ± 9 keV. This value implies a K³⁷ mass excess of -24804 ± 10 keV. Description of a NMR magnetometer and a sixteen-detector array used in conjunction with a 61-cm double-focusing magnetic spectrometer are included in appendices.

Electromagnetic decay of the 1.7- and 2.43-MeV levels in ^9Be

The 1.7- and 2.43-MeV levels in ⁹Be were populated with the reaction ¹¹B(d, α)⁹Be* by bombarding thin boron on carbon foils with 1.7-MeV deuterons. The alpha particles were analyzed in energy with a surface-barrier counter set at the unique kinematically determined angle and the recoiling ⁹Be nuclei at 90^o were analyzed in rigidity with a magnetic spectrometer, in energy by a surface-barrier counter at the spectrometer focus, and in velocity by the time delay between an alpha and a ⁹Be count. When a pulse from the spectrometer counter was in the appropriate delayed coincidence with a pulse from the alpha counter, the two pulses were recorded in a two-dimensional pulse height analyzer. Most of the ⁹Be* decay by particle breakup. Only those that gamma decay are detected by the spectrometer counter. Thus the experiment provides a direct measurement of Γ_rad/Γ. Analysis of 384 observed events gives Γ_rad/Γ = (1.16 ± 0.14) X 10^-4 for the 2.43-MeV level. Combining this ratio with the value of Γ_rad = 0.122 ± 0.015 eV found from inelastic electron scattering gives Γ = (1.05 ± 0.18) keV. For the 1.7-MeV level, an upper limit, Γ_rad/Γ ≤ 2.4 = 10^-5, was determined.

Enzyme induction in Neurospora crassa

I. Studies on Nicotinamide Adenine Dinucleotide Glycohydrase (NADase)

NADase, like tyrosinase and L-amino acid oxidase, is not present in two day old cultures of wild type Neurospora, but it is coinduced with those two enzymes during starvation in phosphate buffer. The induction of NADase, like tyrosinase, is inhibited by puromycin. The induction of all three enzymes is inhibited by actinomycin D. These results suggest that NADase is synthesized de novo during induction as has been shown directly for tyrosinase. NADase induction differs in being inhibited by certain amino acids.

The tyrosinaseless mutant ty-1 contains a non-dialyzable, heat labile inhibitor of NADase. A new mutant, P110A, synthesizes NADase and L-amino acid oxidase while growing. A second strain, pe, fl;cot, makes NADase while growing. Both strains can be induced to make the other enzymes. These two strains prove that the control of these three enzymes is divisible. The strain P110A makes NADase even when grown in the presence of Tween 80. The synthesis of both NADase and L-amino acid oxidase by P110A is suppressed by complete medium. The theory of control of the synthesis of the enzymes is discussed.

II. Studies with EDTA

Neurospora tyrosinase contains copper but, unlike other phenol oxidases, this copper has never been removed reversibly. It was thought that the apo-enzyme might be made in vivo in the absence of copper. Therefore cultures were treated with EDTA to remove copper before the enzyme was induced. Although no apo-tyrosinase was detected, new information on the induction process was obtained.

A treatment of Neurospora with 0.5% EDTA pH 7, inhibits the subsequent induction during starvation in phosphate buffer of tyrosinase, L-amino acid oxidase and NADase. The inhibition of tyrosinase and L-amino acid oxidase induction is completely reversed by adding 5 x 10^-5M CaCl₂, 5 x 10^-4M CuSO₄, and a mixture of L-amino acids (2 x 10^-3M each) to the buffer. Tyrosinase induction is also fully restored by 5 x 10^-4M CaCl₂ and amino acids. As yet NADase has been only partially restored.

The copper probably acts by sequestering EDTA left in the mycelium and may be replaced by nickel. The EDTA apparently removes some calcium from the mycelium, which the added calcium replaces. Magnesium cannot replace calcium. The amino acids probably replace endogenous amino acids lost to the buffer after the EDTA treatment.

The EDTA treatment also increases permeability, thereby increasing the sensitivity of induction to inhibition by actinomycin D and allowing cell contents to be lost to the induction buffer. EDTA treatment also inhibits the uptake of exogenous amino acids and their incorporation into proteins.

The lag period that precedes the first appearance of tyrosinase is demonstrated to be a separate dynamic phase of induction. It requires oxygen. It is inhibited by EDTA, but can be completed after EDTA treatment in the presence of 5 x 10^-5M CaCl₂ alone, although no tyrosinase is synthesized under these conditions.

The time course of induction has an early exponential phase suggesting an autocatalytic mechanism of induction.

The mode of action of EDTA, the process of induction and the kinetics of induction are discussed.